The dynamic ability of tumor cells to adapt to a variety of stresses, including ischemia and cytotoxic chemotherapies, ultimately leads to more aggressive tumor growth and chemoresistance. 14-3-3?, an oncogenic phospho-binding protein, is known to play a central role in this process, yet a fundamental gap exists in our understanding of 1) how 14-3-3? responds to stress to promote cell survival/adaptation; and 2) how 14-3-3? can be targeted to sensitize tumor cells to stress. Until this gap is filled, the therapeutic targeing of 14-3-3? to improve cancer outcomes will be unattainable. The long-term goal is to develop strategies to overcome chemoresistance in cancer and improve patient outcomes. The overall objective of this proposal is to understand a recently discovered 14-3-3?- mediated mechanism of autophagy control and develop strategies to inhibit 14-3-3? in breast cancer. The central hypothesis is that ischemia rearranges the 14-3-3? interactome to promote a ULK1- and AMPK-governed 14-3-3? interaction with phosphorylated Atg9A, which, in turn, promotes autophagy- mediated anthracycline resistance in triple negative breast cancer (TNBC). Additionally, from a therapeutic perspective, it is posited that inhibition of HDAC6, which deacetylates 14-3-3? at critical lysine residues, offers a novel strategy to broadly disrupt 14-3-3? interactions in breas tumors. Guided by strong preliminary data, this hypothesis will be tested in the following specific aims: 1) Determine the mechanism by which ULK1 and AMPK govern Atg9A activity and whether disrupting Atg9A phosphorylation overrides chemoresistance in TNBC; and 2) Target the mechanism of 14-3-3? acetylation to suppress 14-3-3? binding activity in vivo. In the first aim, a combination of proteomics, molecular and microscopy approaches will be used to determine the interplay between AMPK and ULK1 in the regulation of Atg9A phosphorylation and 14-3-3? binding. Additionally, a 14-3-3?-binding defective phosphomutant of Atg9A, which we have already established, will be used to determine whether abrogation of this mechanism blocks anthracycline resistance in TNBC. In aim 2, a clinically approved HDAC6 inhibitor will be tested for its ability to induce 14-3-3? acetylation and disrupt 14-3- 3?-mediated survival pathways in a series of patient derived TNBC xenografts. The approach is innovative because it has utilized 14-3-3? interactomics as a tool to understand adaptive mechanisms of cell survival. Moreover, the approach in aim 2 employs a completely novel strategy to block 14-3-3? in a patient-derived TNBC mouse model. The proposed research is significant because it will contribute fundamentally to our understanding of autophagy, an emerging mechanism of chemoresistance, and could ultimately yield 14-3-3?-targeted strategies to improve clinical outcomes in a patient population (triple negative breast cancer) with limited treatment options.